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Influence of ultrasonic agitation on bond strength,marginal adaptation, and tooth discolorationprovided by three coronary barrier endodonticmaterialsAguiar, Bernardo A; Frota, Luciana M A; Taguatinga, Daniela T; Vivan, Rodrigo R; Camilleri,Josette; Duarte, Marco A H; de Vasconcelos, Bruno CarvalhoDOI:10.1007/s00784-019-02850-y

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Citation for published version (Harvard):Aguiar, BA, Frota, LMA, Taguatinga, DT, Vivan, RR, Camilleri, J, Duarte, MAH & de Vasconcelos, BC 2019,'Influence of ultrasonic agitation on bond strength, marginal adaptation, and tooth discoloration provided by threecoronary barrier endodontic materials', Clinical Oral Investigations, vol. 23, no. 11, pp. 4113-4122.https://doi.org/10.1007/s00784-019-02850-y

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1

Title Page

Authors:

Bernardo A Aguiar DDS1,2, Luciana MA Frota DDS1, Daniela T Taguatinga DDS, MSc3; Rodrigo R Vivan

DDS, MSc, PhD4; Josette Camilleri BChD, MPhil, PhD, FICD, FADM, FIMMM, FHEA5; Marco AH

Duarte DDS, MSc, PhD4; Bruno C Vasconcelos DDS, MSc, PhD1,6

Title:

Influence of Ultrasonic Agitation on Bond Strength, Marginal Adaptation and Tooth Discoloration

Provided by Three Coronary Barrier Endodontic Materials.

Afflitations:

1 Post-Graduate Program in Dentistry, School of Pharmacy, Dentistry and Nursing, Federal University of

Ceará, Fortaleza, Ceará, Brazil. 2 Department of Endodontics, Faculty of Dentistry, University of Fortaleza, Fortaleza, Ceará, Brazil. 3 Endodontist Private Practice, Goiania, GO, Brazil. 4 Department of Dentistry, Endodontics and Dental Materials, Bauru Dental School, University of São

Paulo, Bauru, SP, Brazil 5 School of Dentistry, College of Medical and Dental Sciences, Institute of Clinical Sciences, University of

Birmingham, Birmingham, United Kingdom. 6 School of Dentistry of Sobral, Federal University of Ceará, Campus Sobral, Sobral, CE, Brazil.

Acknowledgement

This study was partially financed by CAPES - Brazilian Federal Agency for Support and Evaluation of

Graduate Education within the Ministry of Education of Brazil.

The authors wish to thank the Analytical Central - UFC / CT - INFRA / MCTI - SISNANO / CAPES Pró-

Equipment for making the analyzes feasible under microscopy.

Corresponding author

Bruno Carvalho de Vasconcelos, DDS, MSc, PhD

Post-Graduate Program in Dentistry, School of Pharmacy, Dentistry and Nursing, Federal University of

Ceará.

1253 Monsenhor Furtado St.

CEP: 60430-355, Fortaleza, CE, Brazil

E-mail: bcv@ufc.br

Manuscript Click here toaccess/download;Manuscript;Manuscript_revised.docx

Click here to view linked References

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ABSTRACT

Objectives: The effect of ultrasonic agitation (UA) on bond strength and adaptation of cervical plugs

prepared with MTA Angelus (MTA), MTA Repair HP (MTAHP) and Biodentine (BIO) was evaluated.

Dentin discoloration caused by the materials/treatment was also assessed. Materials and Methods: Seventy-

two single rooted teeth were divided into six groups depending on the materials/treatment. After cervical

plug preparation, dentin discs were excised for the push-out test; additional discs were analysed under the

confocal microscope to determine adaptation (gaps occurrence). For dentin discoloration analysis (ΔE),

blocks of bovine incisors had cavities prepared and filled with the materials/treatment (from 7 to 180 days).

Results: Both bond strength and adaptation were positively influenced by UA (P<0.05). Comparison

between materials showed an advantage for BIO when compared to MTAHP (P<0.05). The best and worst

results were provided by BIO/UA (12.66 MPa and 1.87%) and MTAHP (2.54 MPa and 28.58%),

respectively. For ΔE, significant differences were observed throughout the periods. Just the MTA without

UA exhibited noticeable discoloration at 180 days (P<0.05). Conclusions: UA favoured a better adaptation

of the materials to the dentin root, resulting in higher bond strength and adaptation of the materials to the

root canal walls. Moreover, UA reduced MTA discoloration, keeping it imperceptible over the period

evaluated. Clinical Relevance: The better adaptation and higher bond strength provided by UA can be

considered clinically relevant due to the importance of maintaining blood clot integrity and the possible

aesthetic compromise provided by reparative materials when used as coronary barrier in regenerative

procedures

Key words: Endodontics, hydraulic calcium silicate cement, marginal adaptation, tooth discoloration,

ultrasonic agitation.

INTRODUCTION

The technique of pulpal revascularization has been considered a promising alternative for

endodontic treatment of teeth with incomplete rhizogenesis and pulp necrosis, since it allows the continuity

of root formation and thickening of the dentin walls [1, 2]. In this technique, a preparation of a coronal

barrier with repairing material is necessary to preserve the integrity of the blood clot and to stimulate the

production of a new tissue capable of re-establishing root formation [3-7].

The mineral trioxide aggregate (MTA) was initially developed to be used as root perforation repair

material [8, 9], however, it has been commonly used as a coronal barrier due to its physico-chemical and

biocompatibility characteristics [6, 9]. However, it presents limitations such as difficulty of insertion and

induction of tooth discoloration [10].

In order to overcome these limitations new materials were developed. Biodentine (Septodont, Saint

Maur des Fosse, Cedex, France) was introduced as a bioactive material. It was based on tricalcium and

dicalcium silicate, main components of Portland cement; commercial MTA materials presents 70-90% of

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Portland cement. Biodentine also presents calcium carbonate and oxides of iron and zirconia, the latter

added as a radiopacifier [10]. This material liquid was composed of water, calcium chloride, added as a

settling accelerator and a water-soluble polymer to improve its plasticity. This cement has adequate

radiopacity, resistance to displacement, short curing time [11, 12], color stability [10], high dimensional

stability and good sealing capacity [13, 14]. Another material developed to overcome the limitations of

conventional MTA is the MTA Repair HP (Angelus, Londrina, PR, Brazil). It is a repair cement based on

tricalcium silicate which also contains additives. According to the manufacturer, these changes provide

better handling, insertion and color stability. The liquid includes an unidentified plasticizer [12, 15]. So far,

the information on the MTA Repair HP is limited; until now its biocompatibility, biomineralization and

resistance to displacement were evaluated showing promising results [12, 15, 16].

In addition to changes in materials composition aiming to improve clinical performance, the

protocols of material insertion were also investigated. The use of ultrasonic indirect agitation was suggested

to improve the sealing ability provided by MTA when used as root-end filling material [17, 18]. The

ultrasonic agitation was also tested directly on filling materials and provided better sealer the penetration

into dentinal tubules [19, 20]. The changes brought by the manufacturers caused a lack of information on

its physical and chemical properties since there is no study under confocal microscopy for the proper

evaluation of the marginal adaptation.

Moreover, considering the use of these materials in the aesthetic area at the cervical level and

considering that the MTA presents notably the capacity to induce the dentin discoloration, it would be

relevant to evaluate the influence of this event by the radiopacifiers of the new cements. As well as, the use

of the ultrasonic agitation in the evaluation of the adaptation would be interesting to evaluate its effect in

the darkening.

Considering the novel materials disposable for coronal barrier preparation, the possible influence

of ultrasonic agitation on its adaptation and the parallel risk of tooth discoloration, the current study

evaluated the influence of ultrasonic agitation on push-out bond strength and marginal adaptation to root

canal walls in the cervical region of MTA Angelus (Angelus), MTA Repair HP and Biodentine; also

evaluated the tooth discoloration promoted by these materials. It will be considered as null hypotheses the

absence of differences in bond strength, marginal adaptation, and tooth discoloration provided by the

materials in function of the use or not of the ultrasonic agitation.

MATERIALS AND METHODS

Bond strength tests and marginal adaptation

The sample calculation was performed using the G*Power v3.1 for Mac (Heinrich Heine,

Universität Düsseldorf) by selecting the Anova One Way fixed test. Data from an earlier study were

considered and the effect of sample size in the present study was established [20]. The effect size in the

present study was established (=1.36) and the alpha type error of 0.05 in the beta power of 0.80. A total of

9 samples were indicated as the ideal size to observe significant differences; the sample was increased by

30% for safety if specimens were to be lost (n = 12).

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Selection and preparation of the sample

After approval by the Local Ethics Committee (#1.900.129/2017), 72 maxillary uni-radicular

human teeth extracted for unknown reasons were collected for the studies of bond resistance and marginal

adaptation. Coronary access with diamond tips (#1014 and #3081; KG Sorensen, Cotia, SP, Brazil) was

carried out, followed by preparation of the cervical and middle thirds with Largo Reamer #5 (Dentsply-

Sirona, Ballaigues, Switzerland). After the preparation specimens were evaluated for circularity and

standardization of the cavity diameter. Where the Largo Reamer #5 did not enter just the same has been

replaced. The teeth were then cross-sectioned 5.0 mm below the cemento-enamel junction by means of a

precision cut double-face diamond disk (Isomet 1000; Buhler Ltda., Lake Bluff, NY, USA). After being

sectioned, the teeth were submitted to ultrasonic baths with 2.5% sodium hypochlorite solution (Asfer

Indústria Química Ltda, São Caetano do Sul, SP, Brazil) for 15 minutes, with distilled water (Asfer Indústria

Química Ltda.) for 1 minute, with 17% ethylenediaminetetraacetic acid (EDTA) (Biodinâmica Química e

Farmacêutica Ltda., Ibiporã, PR, Brazil) for 3 minutes and finally with distilled water for 3 minutes.

After specimen preparation, collagen sponge plugs (Technew, Rio de Janeiro, RJ, Brazil) were

inserted into the canal with the aid of a Schilder condenser (Odous de Deus, Belo Horizonte, MG, Brazil)

obliterating the apical 2 mm. Regardless of the group in which they were to be allocated, the specimens

were irrigated with 1 mL of distilled water. Afterwards, gentle aspiration was performed with the aid of the

endodontic aspirator for 4 seconds and additional drying with absorbent paper cones (Dentsply-Sirona).

Manipulation and insertion of materials

The manipulation of the repair cements was carried out by a single operator previously calibrated,

following the manufacturer's instructions; Fluo-3 fluorophore (Sigma-Aldrich, Warrington, PA, USA) at a

concentration of approximately 0.1% (w/w) was incorporated into the materials to determine marginal

adaptation [21].

The specimens were initially randomly assigned to three experimental groups, depending on the

material (n = 24). For insertion of the materials a 1.2 mm diameter MTA microapplicator (Angelus) was

used followed by condensation with the Schilder condenser. The procedure was repeated until cervical

plugs reached 3.0 mm thickness. Then, the specimens were randomly divided again between the subgroups

with and without ultrasonic agitation (UA) (n = 12). In the groups which received ultrasonic agitation

(MTA/UA, MTAHP/UA and BIO/UA), piezoelectric ultrasound equipment (Piezon Master 200; EMS,

Nyon, Geneva, Switzerland) was set to 10% of power. A specific ultrasonic tip (E5; Helse Ultrassonic,

Santa Rosa do Viterbo, SP, Brazil) was inserted into the material at a depth of 2.0 mm. Two 20-second

activation cycles were performed, one in the mesio-distal direction and one in the vestibular-palatine

direction [19]. Then, a final vertical condensation was performed to accommodate the materials; similarly,

the specimens allocated in groups without UA also received an additional vertical condensation.

After concluding the cervical plugs, temporary restorations with glass ionomer cement (GIC; FGM

Produtos Odontológicos Ltda, Joinville, SC, Brazil) were performed in order to simulate clinical

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procedures. The specimens were then stored with the apical portions immersed in phosphate-buffered saline

solution (PBS; Quimlab, Jacareí, SP, Brazil) in an oven at 37 °C for 7 days.

After the storage period, two 1.0 mm thickness dentin discs were extracted from each plug on its

central portion using a cutting machine (Isomet 1000; Buhler Ltda., Lake Bluff, NY, USA) totalling 144

discs; each disk obtained were allocated to one of the tests proposed - the cervical one for the bond strength

test and the apical one for the marginal adaptation test - the portion directly in contact with GIC was

discharged in order to allow any interference.

Evaluation of the bond strength (push-out)

The 72 dentin disks allocated for the bond strength test were tested using a Universal Testing

Machine (#443; Instron, Norwood, MA, USA) handled by a blind operator; for performing the push-out

test using a constant velocity of 0.5 mm/min. For this, punches with 1.2 mm diameter were positioned in

the center of the upper face of the plugs; on the underside, a steel plate with a 3.0 mm diameter central bore

was adapted to serve as a support for the specimen and enable material to escape from the plugs. The

maximum force required for cement displacement was measured in Newtons (N) and converted to MPa by

the division of force (N) by the area in mm2 (1MPa = 1N/mm2).

For analysis of fracture types after the push-out test, all slices were examined in a scanning electron

microscope (Aspex Express; FEI Europe, Eindhoven, Netherlands) at a magnification of 30x. The failures

were classified as: adhesive - rupture of the union at the interface dentin/material, cohesive - rupture on the

material and, mixed – rupture cohesive of the material and adhesive in the dentin.

Assessment of marginal adaptation

The remaining 72 discs were adapted to glass slides and fixed with wax with the coronary face

facing upwards. The sets were taken to the politriz (Arotec, Cotia, SP, Brazil) where, using 600, 900 and

1200 granulometry water sands, were polished. Then the samples were taken to Confocal Laser Scanning

Microscope (LSM 710; Carl Zeiss Microscopy GmbH, Baden-Württemberg, Germany) handled by a blind

operator and examined 10 μm below the surface under 20x magnification; 10 section scans of 1 μm were

recorded at a resolution of 1024 x 1024 pixels using the ZEN 2012 program (Carl Zeiss Microscopy GmbH)

and saved in TIFF format.

To measure the adaptation (i.e. presence of gaps), the images were evaluated in the Image J

software (National Institute of Health, Bethesda, MD, USA), using as a calibration tool the scale offered

by confocal microscopy images. The total perimeter of the canals and the length of gaps were determined

in millimetres using the software polygonal and linear tools, respectively; once these measurements were

obtained, the percentages of gaps were calculated according to the total perimeter of the root canals.

Study of tooth discoloration

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Before performing this evaluation, a sample size calculation was performed following the same

parameters used in the previous test; based on the preliminary study results the test pointed to a sample of

10 specimens as a sufficient sample to determine possible statistical differences [22]. An experimental

design of this study methods was show in Figure 1.

Selection and preparation of the sample

For the analysis of dentinal discoloration, bovine incisor blocks prepared according to the model

proposed by Marciano et al. [22]. Sixty-five blocks (10 mm x 10 mm) were prepared with the aid of a

diamond blade driven by a cutting machine. After being prepared, they had their lingual faces sectioned in

manner to expose their pulp chambers. Cavities 5 mm in diameter and approximately 1.5 mm deep were

prepared in the center of the lingual face of each specimen using diamond tips #4054 (KG Sorensen);

specimens where a remainder of 2 ± 0.2 mm of dentin and enamel thickness were not obtained at the bottom

of the cavity were replaced.

Subsequently, the specimens were submitted to ultrasonic baths following the previously used

protocol. Afterwards, after being dried with filter paper, the wells had their external boundary conditioned

with 37% phosphoric acid for 15 seconds, washed with distilled water for 1 minute and gently air-dried for

15 seconds. An adhesive layer (Adper Single Bond 2; 3M ESPE, Sumaré, SP, Brazil) was applied in the

conditioned and polymerized area (Optilight LD Max; Gnatus, Ribeirão Preto, SP, Brazil) for 20 seconds

to allow interface resin.

Materials manipulation and insertion

After specimen preparation, they were randomly divided among the six experimental groups (n =

10). The cements, after being manipulated, were inserted into the cavities. For those who underwent

ultrasonic agitation (MTA/UA, MTAHP/UA and BIO/UA), it was accomplished by means of the use of

piezoelectric ultrasound programmed for power 3 (approximately 30%), with a smooth tapered tip (E5;

Helse Ultrasonic). After gentle condensation of the cements in the cavities, the tip was inserted at a depth

of 1 mm. Then, two cycles of agitation of 20 seconds, one in the mesio-distal and one in the buccal-palatal

direction were performed [19]; independent of the group to which they took part, the materials were once

again condensed to fill the entire cavity.

After completion, restorations were performed with flow resin (New DFL, Rio de Janeiro, RJ,

Brazil), which was polymerized with LED light (Optilight LD Max, Gnatus) for 60 seconds. The remaining

5 blocks comprised the negative control group in which the cavities were completely filled with restorative

material. Regardless of the group, the specimens were immersed in individual containers containing 2 mL

of distilled water, where they remained throughout the experimental period at 37ºC and 100% humidity.

Evaluation of tooth discoloration

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Color determination was performed using a digital spectrophotometer (VITA Easyshade compact;

VITA Zahnfabrik AG, Bad Sachington, Germany). The equipment was calibrated prior to the measurement

of each specimen. Measurements were obtained immediately after placement of the materials (reference

color; 0) after 7, 14, 30 and 180 days. Color parameters were recorded as determined by the International

Commission On Illumination (CIE, 1978), considering "L", "a" and "b", where "L" represents the values of

color luminosity, "a" corresponds measurement along the red-green axis and "b" is the measurement along

the yellow-blue axis. The change in color (ΔE), in relation to time intervals, was calculated based on the

initial values using the following formula:

ΔE = [(L1-L0)2 + (a1-a0)2 + (b1-b0)2]1/2

Statistical analysis

The data of the evaluations were tabulated and tested for their normality by the Shapiro-Wilk test,

which indicated a parametric nature for the bond strength and dentin discoloration and non-parametric data

for the analysis of the adaptation to the canal walls. Thus, ANOVA and Tukey tests were used for the bond

strength and discoloration tests, and Kruskal-Wallis and Dunn tests were used for analysis of adaptation.

In addition, the influence of ultrasonic agitation was statistically tested in all evaluations, regardless of the

material used, and Student's t-test and Mann-Whitney tests, respectively. For all comparisons, the

significance levels were established at 5%.

Results

Bond strength

Table 1 presents the results of the push-out test. Premature failures were not observed in any

groups/subgroups. The BIO/UA group exhibited the best bond strengths while the MTAHP group without

agitation had the worst. Considering each material with and without treatment, statistically significant

differences were observed between the BIO groups; the UA positively influenced their bond strength

(P<0.05). Comparing the materials submitted or not to UA, significant differences were observed in both

conditions, with MTAHP offering significantly lower results than the others (P<0.05); no differences were

observed between BIO and MTA. Considering only the use or not of UA, significant differences were

observed, where the UA proportioned superior results (P<0.05). The MTA/UA and BIO groups, with and

without UA, presented a predominance of the cohesive failures, and the MTAHP group presented

predominance of the cohesive type as could be observed in Table 1; Figure 2 presents the failure types

illustration.

Analysis of the interface of adaptation to the dentin wall

Percentage of gaps observed in the sections of the cervical plugs prepared with the repair materials

with or without UA are shown in Table 2. Once more the BIO/UA exhibited the optimal interface with

adequate marginal adaptation, giving only 1,87% of gaps. The worst results were exhibited by MTAHP

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without UA which presented 28.58% of maladaptation. The comparisons between groups on both materials

and treatment did not show significant differences (P>0.05). However, isolating the variables, significant

difference was observed between BIO and MTAHP (P<0.05). A significant difference was also observed

when only the treatment was considered, with UA increasing the adaptation of the materials to the root

canal walls (P<0.05). Figure 3 shows representative images of specimens from the experimental groups

obtained by confocal microscopy.

Analysis of tooth discoloration

The specimens of the control group exhibited no color change (ΔE). The data relating to ΔE for

the experimental groups are shown in Table 3. The MTA specimens were the only ones to show

discoloration above the clinically perceptible (ΔE ≥ 3.7). This occurred after 7 days of observation. Between

15 and 180 days it presented stability, offering the maximum of ΔE at 30 days (7,16). Significant differences

between the MTA and the other groups were observed in all evaluated periods (P<0.05); the BIO and

MTAHP/UA groups took turns as the best results. Representative images of the specimens from each group

can be seen in Figure 4.

Discussion

The materials used were quite similar in composition and were all based on tricalcium silicate with

similar hydration reaction. The MTA Angelus is a first-generation MTA with slight modifications. The

Biodentine and MTA Repair HP are no longer based on Portland cement but on laboratory synthesized

calcium silicates, both still include additives such as calcium carbonate, calcium oxide and polymer that

enhance the material performance. The radiopacifiers are also different for the materials tested. The MTA

Angelus has retained the bismuth oxide of the original formulation while Biodentine and MTA Repair HP

include alternative radiopacifiers namely zirconium oxide and calcium tungstate, respectively. Although

these materials have already been tested as retro plugs. This simple fact does not respond to possible

differences when used as cervical plugs by the aspect of cavity size in relation to agitation / volume of

material and aesthetic by use at the cervical level. Considering the results obtained, the null hypothesis

suggested was rejected since the ultrasonic agitation favoured better bond strength and marginal adaptation

of the materials to the canal walls and altered the tooth discoloration exhibited by the MTA Angelus.

The methods used closely simulated the clinical conditions and the protocols employed were based

on the suggestions of the American Association of Endodontists, especially regarding the coronary barrier

preparation for regenerative procedures [4]. Still, simulating teeth with incomplete rhizogenesis, the

circularity and the cavity diameter were standardized with a Largo Reamer #5 (Dentsply-Sirona). When a

restorative material is used, one of its most desirable property is sealing, therefore, it must withstand the

forces that may cause its displacement, such as functional pressures, or the application of other restorative

materials [12]. In this way, the push-out test has been used as an efficient and feasible method to evaluate

the strength of bond strength between dentin/material and material/material [11, 12]. The protocol used for

this type of evaluation has been widely described in the literature [11, 12, 20].

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The marginal adaptation is a characteristic which is closely related to the bond strength. For this

evaluation, a confocal laser scanning microscope was used as employed in large number of previous studies

[19, 23, 24]. Previously, a pilot study was carried out not finding differences in the characteristics of the

cements when using the flurophore, in agreement with Wiesse et al. [20]. The fluorophore Fluo3 was the

chosen fluorescence indicator. Fluo-3 take the place of the commonly employed Rhodamine B once it binds

to the calcium ions, which is an element present in the great quantity in the materials based on tricalcium

silicate, as pointed out by Jeong et al. [24].

The tooth discoloration was investigated using methods previously described in the literature [25-

27]. Even recognizing that the dental substrate employed is not exactly the same used in bond strength and

adaptation analyzes, it should be considered that it presents advantages in function of, as opposed to human

teeth that have a smaller surface and usually have restorations or caries that can interfere in the color

analysis, the bovine teeth provide a larger flat surface area, allowing adequate and standardized color

evaluation [26]. In addition, bovine teeth are similar to human teeth in the composition of type I collagen

of the organic matrix, which accredits the method for microstructural issues considering that the interaction

between radiopacifiers and this protein are blamed for the darkening process [22, 26-28].

Ultrasonic agitation is based on the transmission of micrometric acoustic energy. Such mechanical

vibration energy is expended from the insert and transmitted to the material, favouring a greater penetration

into the dentinal tubules and a better adaptation of cement/dentin interface [19, 20, 29]. In the present study,

bond strength and improvement in dentin adaptation values were observed when ultrasonic agitation was

used. It is postulated that ultrasonic agitation produced an increase of the cement pressure against the canal

walls favouring a more effective filling of the irregularities, improving adaptation to root canal walls and,

consequently, their bonding resistance [19]. Considering this observation, it could be suggested that this

better adaptation increases the materials adhesive strength making it superior than its cohesive strength.

Additionally, evaluating the role of the materials independently of the treatment, Biodentine

presented results of bond strength and adaptation to the dentine significantly superior to those provided by

the MTA Repair HP. It is suggested that the results obtained by Biodentine can be attributed to the more

plastic consistency of the material [20, 30].

Regarding dentinal discoloration, the results found in the present study confirms the color stability

of the teeth treated with Biodentine and MTA Repair HP, corroborating with findings from previous studies

that pointed the color stability of the first material [27, 31]. To date there was no information on the

discoloration if any caused by MTA Repair HP. Knowledge of the discoloration mechanisms and it being

attributed to the presence of bismuth oxide in materials, the findings in this study were as anticipated. The

materials that were bismuth oxide free did not discolour. The novelty lies with the effect of ultrasonic

agitation on the discoloration where use of ultrasonic agitation mitigated the discoloration caused by MTA

Angelus. Thus, ultrasonic agitation can be suggested as another way of reducing the bismuth oxide induced

discoloration. The dentin discoloration caused by the bismuth oxide present in MTA Angelus and its

interaction with the dental structures had already been reported [22, 27, 28]. This finding was also observed

in the present study, being this the only material to produce a color change higher than 3.7, an index that

was perceived as being perceptible to the human eye [32]. However, results of the present study indicate

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that ultrasonic agitation is capable of positively influencing the color stability of bismuth oxide,

consequently of MTA Angelus, an aspect highlighted by the statistically significant difference observed at

15 and 180 days between the MTA groups with and without agitation. Yet, in the observed periods the

ultrasonically agitated MTA Angelus did not show color alteration higher than the perceptible values. It is

known that discoloration caused by bismuth oxide results during the hydration process in its interaction

with strong oxidants such as the amino acids present in the dentin collagen [22]. It is believed that this

finding may be related to the reduction of water available for hydration - the result of the warming generated

by the ultrasound action, reducing the number of particles reacted with the collagen matrix. However, other

investigations are necessary to prove these hypotheses.

Considering the importance of maintaining blood clot integrity and the possible aesthetic

compromise provided by reparative materials when used as coronary barrier in regenerative procedures,

the findings of the present study suggest that ultrasonic agitation can improve the resistance to

displacement of materials and their marginal adaptation; yet, positively influence the color stability of

the MTA Angelus. Thus, although more studies are necessary to confirm the hypotheses raised and to

evaluate other possible interactions, it can be indicated that the use of ultrasonic agitation during the

revascularization procedures presents itself as an accessible and feasible tool for clinical use, capable of

increasing the quality of the procedures performed.

Conclusion

It was concluded that the repair materials evaluated were unable to provide total adaptation to

the root canal walls, however, the use of ultrasonic agitation provided better bond strength and marginal

adaptation of these. Furthermore, the Biodentine and MTA Repair HP cements did not cause dentine

discoloration, however the white MTA Angelus promoted it, having its effect reduced by the use of

ultrasonic agitation.

Compliance with Ethical Standards

Conflict of Interest: The authors deny any conflicts of interest related to this study.

Funding: This study was partially financed by CAPES - Brazilian Federal Agency for Support and

Evaluation of Graduate Education within the Ministry of Education of Brazil.

Ethical approval: This study was previously approved by Local Ethics Committee (#1.900.129/2017) and

were in accordance with the ethical standards of the institutional and/or national research committee and

with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent: Informed consent was obtained from all individual participants included in the study.

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Figures and legends

Figure 1. Experimental design of discoloration analysis methods.

Figure 2. Representative images of failure types observed; A. Cohesive failure (Biodentine with ultrasonic

agitation), B. Adhesive failure (MTA Repair HP without ultrasonic agitation), and C. Mixed failure (MTA

Angelus withou ultrasonic agitation).

Figure 3. Confocal microscopy of the perimeter of gaps at the interface of the cervical dentin with the

repairing material using Fluo-3 with wavelength of 506 nm of excitation and 526 nm of emission. Images

made at 10 μm below the surface of the sample with a magnification of 20x. 10 sections of 1μm were

obtained. Images recorded in CZI with resolution of 1024 x 1024 pixels using the program ZEN 2012 (Carl

Zeiss Microscopy GmbH, Baden-Württemberg, Germany) and saved in TIFF format. Measurement of

adaptation defects at the cement/dentin interface (gaps) in the Image J program (National Institute of Health,

Bethesda, MD, USA) with 100 μm scale.

Figure 4. Representation of bovine teeth samples filled with MTA Angelus, MTA Repair HP and

Biodentine as a function of ultrasonic agitation. The discoloration is evident in the MTA Angelus group

without using the ultrasound with color change of the peripheral dentine to the material. The other groups

do not show any color changes.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Figure 1 Click here to access/download;Figure;Figure_1.jpeg

Table 1. Bonding strength (MPa) of restorative materials to root dentin as a function of ultrasonic

agitation and distribution of failure types (%).

Material Agitation Bond strength Failure type

Mean ± sd Adhesive Coesive Mixed

MTA-Angelus No 6.83 ± 1.99b,c 31.25 12.5 56.25

Yes 9.54 ± 3.35a,b 12.5 68.75 18.75

MTA Repair HP No 2.54 ± 1.26d 56.25 37.5 6.25

Yes 4.13 ± 2.43c,d 25 25 50

Biodentine No 5.86 ± 2.55c 6.25 68.75 25

Yes 12.66 ± 5.92a 6.25 68.75 25

a,bDifferent lowercase letters overlapped represent significant differences between the groups of material

according to the ANOVA and Tukey tests (P <0.05).

Table 1

Figure 2 Click here to access/download;Figure;Figure_2.jpg

Figure 3 Click here to access/download;Figure;Figure_3.jpg

Figure 4 Click here to access/download;Figure;Figure_4.jpg

Table 2. Percentage of gaps in relation to the perimeter provided

by the repair materials as a function of the ultrasonic agitation.

Material Agitation Percentage of gaps

Median (min. - max.)

MTA-Angelus No 11.92 (0.0 – 48.65)a,A

Yes 5.34 (0.0 – 65.47)a,A

MTA Repair HP No 28.58 (8.01 – 63.73)a,A

Yes 17.87 (0.0 – 43.26)a,A

Biodentine No 10.14 (0.0 – 27.56)a,A

Yes 1.87 (0.0 – 16.33)a,A

a,bDifferent lowercase letters overlapped represent significant

differences between the groups according to the materials according to

the Kruskal-Wallis and Dunn tests (P <0.05).

Table 2

Table 3. Mean and standard deviation of the discoloration analysis provided by the repair materials

as a function of ultrasonic agitation.

Material Agitation

7 days 15 days 30 days 180 days

Mean ± sd Mean ± sd Mean ± sd Mean ± sd

MTA-Angelus No 4.91 ± 2.85b 6.05 ± 4.05b 7.16 ± 3.81b 6.10 ± 1.79b

Yes 2.11 ± 0.97a 4.09 ± 1.64ab 2.59 ± 1.36ab 2.68 ± 1.18a

MTA Repair HP No 2.77 ± 1.12ab 2.70 ± 0.74ab 3.26 ± 1.47ab 2.60 ± 0.79a

Yes 1.95 ± 1.38a 1.75 ± 1.46a 1.44 ± 1.33a 1.68 ± 0.67a

Biodentine No 1.86 ± 0.87a 2.04 ± 0.68a 2.72 ± 0.86ab 2.33 ± 0.71a

Yes 2.35 ± 0.36ab 2.61 ± 0.99ab 3.39 ± 0.67ab 2.25 ± 1.36a

a,bDifferent lowercase letters overlapped represent significant differences between groups, considering each

evaluation period, according to the ANOVA and Tukey tests (P <0.05).

Table 3

AUTHOR’S RESPONSE TO EDITOR/REVIEWERS

Dear Prof. Imad About, Ph.D.

Associate Editor of the Clinical Oral Investigations.

Thank you for allowing us to make the changes suggested by the Reviewers in order to qualify

this paper for possible publication in the Clinical Oral Investigations.

Below are some answers to the Reviewers comments:

Reply to Reviewers # 1

Initially thanks for your considerations and your commendation. Our response for your

concerns are listed below:

1. Concern of the Reviewer: Methods: What did the authors use for root canal preparation? diamond

bur, gates glidden or lentulo?? Please see page 8-line 46 and page 4-line 8. Is not it difficult to insert

the diamond bur up to the middle third of the root? I think, this part is a bit confusing.

Our response: For cervical and middle thirds preparation a Largo Peeso Reamer #5 (Dentsply-Sirona,

Ballaigues, Switzerland) was used in long axis of the teeth. Please, apologies for this typing error.

Revised text: Page 4, Lines 8 and 11: …was carried out, followed by preparation of the cervical and middle thirds

with Largo Reamer #5 (Dentsply-Sirona, Ballaigues, Switzerland). After the preparation specimens

were evaluated for circularity and standardization of the cavity diameter. Where the Largo Reamer #5

did not enter…

Page 8, Line 52: … and the cavity diameter were standardized with a Largo Reamer #5 (Dentsply-

Sirona).

2. Concern of the Reviewer: Table 1-Table 3: I suggest giving mean and standard deviation values in

one column in order to make table simple. Please see following examples;

mean ± sd

6.83± 1.99bc

For Table 2:

median (min - max)

11.92 (0 - 48.65) a,A

Our response: Following the guidance of the Reviewers the alterations was performed in Tables 1-3.

Revised text:

Revisions were highlighted in Tables 1-3.

3. Concern of the Reviewer: Could you please insert a scale bar on confocal images? Additionally,

the way to be outlined the region of interest (ROI) using the polygonal tool in one of the sections could

be stated if possible.

Our response: Regards of the scale bar, it was inserted in Confocal images at Figure 3 (Figure 1 in

original submission). With respect to the inclusion of measurement procedures performed in Image J

we do not understand that an illustration of it in Figure 3 was indicated; we think that it could turn

difficult images comprehension and group comparisons. Whereas, a text better explaining which were

Image J tools used was included in the text.

Revised text:

Authors' Response to Reviewers' Comments Click here to access/download;Authors' Response toReviewers' Comments;Point-to-

Page 5, Line 53: The total perimeter of the canals and the length of gaps were determined in

millimetres using the software polygonal and linear tools, respectively.

4. Concern of the Reviewer: Did you observe premature failure during push-out bond strength test?

Our response: No premature failures were observed during sample preparation for the push-out bond

strength test or during it. Considering this an important information a sentence stating it was inserted in

Results chapter.

Revised text:

Page 7, Line 36 and 37: Table 1 presents the results of the push-out test. Premature failures were not

observed in any groups/subgroups. The BIO/UA…

5. Concern of the Reviewer: I suggest adding a photograph of representative dentin discs showing

cohesive failure or other failure types, if it is available. Furthermore, authors could state the percentage

of failure types for each group (page 7-line 45).

Our response: Considering the asked revision, the percentages of failures types were inserted in Table

1 for better comprehension. Related to the failure types illustration, images were included (Figure 2).

Revised text: Following the guidance of the Reviewers Table 1 was improved.

Page 7, Line 49 to 51: The MTA/UA and BIO groups, with and without UA, presented a predominance

of the cohesive failures, and the MTAHP group presented predominance of the cohesive type as could

be observed in Table 1. Figure 2 presents the failure types illustration.

6. Concern of the Reviewer: Discussion: Please improve discussion with the explanation of why most

of the groups exhibited cohesive type of failure.

Our response: Related to this point, observed in UA and Biodentine groups, we understand that the

cohesive failures observed predominantly in the groups under ultrasonic agitation could be provided by

material better adaptation to the root canal walls. This adaptation increases the adhesive strength

making it superior than the cohesive strength of the materials.

Revised text: Page 9, Line 29 to 35: It is postulated that ultrasonic agitation produced an increase of the cement

pressure against the canal walls favouring a more effective filling of the irregularities, improving

adaptation to root canal walls and, consequently, their bonding resistance [19]. Considering this

observation, it could be suggested that this better adaptation increases the materials adhesive strength

making it superior than its cohesive strength.

7. Concern of the Reviewer: Are doi addresses necessary? Please check the journal reference style.

Following is an example of the reference of journal article;

Smith J, Jones M Jr, Houghton L et al (1999) Future of health insurance. N Engl J Med 965:325-329.

Our response: Taking account the Instruction for Authors accessed in December 26th the most

complete example available is:

Journal article:

Gamelin FX, Baquet G, Berthoin S, Thevenet D, Nourry C, Nottin S, Bosquet L (2009) Effect of high

intensity intermittent training on heart rate variability in prepubescent children. Eur J Appl Physiol

105:731-738. https://doi.org/10.1007/s00421-008-0955-8

Thus, considering the Instructions we think that was indicated to state the DOI number for all listed

References.

Revised text:

Not applicable.

Reply to Reviewer # 2

Thanks for considering this study clinically relevant and to permit us to explain our typing and

methodological options.

1. Concern of the Reviewer: My main major comment is: why didn't researchers consider this study

in two separate parts (adhesive/interface analysis and discoloration). Too many parameters in one paper

made the text confusing.

Our response: Honestly, at first, we thought of writing two papers considering the differences between

the methodologies employed. However, when we started writing the first of them, we understood that,

in clinical terms, one analysis could reinforce or compromise the other. We believe that it would be

ambiguous to conclude that the ultrasonic agitation would be favorable in terms of adaptation and bond

strength if it would compromise the color stability of the materials. Based on the above, we chose to

write a single paper, even more complex, but more complete and with greater clinical impact and

citation potential. We hope that this explanation could make sense, in any case, we are open to

suggestions.

Revised text:

Not applicable.

2. Concern of the Reviewer: Furthermore, one part used extracted teeth, the second part used bovine

teeth. The first part: the method is understandable; the second part (discoloration part) needs a

schematic drawing to be able to make the method understandable.

Our response: We agree the comprehension improvement that an Experimental Design could produce

in method description of Discoloration Evaluation. Thus, an image presenting it sequence was included

(Figure 1).

Revised text:

Not applicable.

3. Concern of the Reviewer: If the authors could divide the paper into two separate parts, they could

add SEM pictures from the failed samples.

Our response: This point was also commented by the Reviewer #1; representative images of the

failure types were now present in paper as Figure 2.

Revised text: Not applicable.

4. Concern of the Reviewers: …could make a more specific discussion and could give more

knowledge.

Our response: We understand the point of view highlighted by the Reviewer and tried to better

separate the Discussion related to each studied subject. Some information was really mixed in the

original submission. We think that after corrections and inclusion asked during this review that

confusion was mitigated.

Revised text:

Not applicable, spread throughout the discussion chapter.

5. Concern of the Reviewers: Abstract: needs revision. Method does not include the details of the

study groups;

Our response: During this paper preparation, the most difficult part to write was the Abstract, mainly

considering the recommendation in Authors Instructions to respect the number of 250 words. Thus,

considering the word limitation we opted to describe the groups in the Objective section, it was stated

as: “Objectives: The effect of ultrasonic agitation (UA) on bond strength and adaptation of cervical

plugs prepared with MTA-Angelus (MTA), MTA Repair HP (MTAHP) and Biodentine (BIO) was

evaluated.” – it intends to presents MTA, MTA/UA, MTAHP, MTAHP/UA, BIO, and BIO/UA. We

wish to better describe this group division; however, the word limitation makes such inclusion almost

impossible.

Revised text: Not applicable.

6. Concern of the Reviewer: Discussion: needs discussion of the differences between the test samples

(bovine and human teeth).

Our response: As requested by the Reviewer an extended Discussion about the method option for

bovine blocks and its advantages was inserted in the text.

Revised text:

Page 9, Line 12 to 22: The tooth discoloration was investigated using methods previously described in

the literature [25-27]. Even recognizing that the dental substrate employed is not exactly the same used

in bond strength and adaptation analyzes, it should be considered that it presents advantages in function

of, as opposed to human teeth that have a smaller surface and usually have restorations or caries that

can interfere in the color analysis, the bovine teeth provide a larger flat surface area, allowing adequate

and standardized color evaluation [26]. In addition, bovine teeth are similar to human teeth in the

composition of type I collagen of the organic matrix, which accredits the method for microstructural

issues considering that the interaction between radiopacifiers and this protein are blamed for the

darkening process [22, 26-28].